WO2009155637A1 - Protocols for treating and preventing obesity and complications arising therefrom - Google Patents

Protocols for treating and preventing obesity and complications arising therefrom Download PDF

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Publication number
WO2009155637A1
WO2009155637A1 PCT/AU2009/000776 AU2009000776W WO2009155637A1 WO 2009155637 A1 WO2009155637 A1 WO 2009155637A1 AU 2009000776 W AU2009000776 W AU 2009000776W WO 2009155637 A1 WO2009155637 A1 WO 2009155637A1
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Prior art keywords
whey
adipogenesis
fraction
casein
glycoproteins
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PCT/AU2009/000776
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English (en)
French (fr)
Inventor
Antonio Rajic
Dominic J Autelitano
Ana K Vrkic
Christopher G Hosking
Leodevico L Ilag
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Healthlinx Limited
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Priority claimed from AU2008903257A external-priority patent/AU2008903257A0/en
Application filed by Healthlinx Limited filed Critical Healthlinx Limited
Priority to US13/000,104 priority Critical patent/US20110165138A1/en
Priority to AU2009262338A priority patent/AU2009262338A1/en
Priority to EP09768610A priority patent/EP2318021A4/de
Publication of WO2009155637A1 publication Critical patent/WO2009155637A1/en
Priority to US14/453,017 priority patent/US20140348812A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/20—Milk; Whey; Colostrum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
    • A61K38/018—Hydrolysed proteins; Derivatives thereof from animals from milk
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates generally to the field of obesity and complications arising therefrom and in particular to the control of obesity by inhibiting adipogenesis and related processes.
  • Adipogenesis is defined as the formation of fat or fatty tissue, the development of fat cells from pre-adipocytes, has been one of the most intensely studied models of cellular differentiation. In part this has been due to the availability of in vitro models which recapitulate most of the critical aspects of fat cell formation in vivo.
  • the mouse 3T3-L1 preadipocyte cell line is one of the best characterised models of adipocyte differentiation.
  • induced preadipocytes undergo a post-confluent mitosis at about 24 hours and subsequent growth arrest that commits the cell to differentiation to an adipocyte phenotype. Alterations in the regulation and expression of as many as 300 proteins may be associated with the process of preadipocyte differentiation.
  • Activation of sterol regulatory binding proteins (SREBPs) and repression of AP-2a and SPl transcriptional activity are also considered to be crucial early events in adipocyte differentiation (Smas and SuI, 1995 supra; Morrison and Framer, 2000 supra).
  • PPAR ⁇ and C/EBP ⁇ in particular are considered to be crucial in regulating genes involved in the maturation of the adipocyte phenotype (Smas and SuI, 1995 supra; Morrison and Framer, 2000 supra; Ntambi and Kim, 2000 supra). Late markers of differentiation including adipocyte specific enzymes involved in lipid metabolism begin to be expressed by day 3 and terminally differentiated adipocytes containing obvious cytoplasmic lipid droplets are evident by days 5-7 (Smas and SuI, 1995 supra; Ntambi and Kim, 2000 supra).
  • the hormonal induction of preadipocyte differentiation can be mediated by a variety of factors including insulin which acts via the insulin-like growth factor- 1 (IGF-I) receptor, corticosteroids acting via the glucocorticoid receptor and by various agents that activate the cAMP-dependent protein kinase pathway (Smas and SuI, 1995 supra; Morrison and Framer, 2000 supra; Ntambi and Kim, 2000 supra). Inhibition of these pathways and/or their downstream effectors such as transcription factors PPAR ⁇ and C/EBP ⁇ thus provide potential targets for modulating adipocyte differentiation and maturation.
  • IGF-I insulin-like growth factor- 1
  • TGF ⁇ tumour necrosis factor ⁇
  • EGF epidermal growth factor
  • bFGF basic fibroblast growth factor
  • TGF ⁇ transforming growth factor ⁇
  • activators of protein kinase C and calcium ionophores can lead to rapid down regulation of PP AR ⁇ and inhibition of mature adipocyte differentiation (Smas and SuI, 1995 supra; Xing et al, Endocrinology 138(7):2776-2783, 1997).
  • preadipocyte factor- 1 (pref-1) Down regulation of the EGF repeat domain-containing transmembrane protein preadipocyte factor- 1 (pref-1) in mature adipocytes has been shown to be associated with preadipocyte differentiation and drugs such as statins which induce high pref-1 levels have been shown to inhibit adipocyte differentiation (Nicholson et al, Br. J. Pharmacol. 757:807-815, 2007).
  • statins which induce high pref-1 levels have been shown to inhibit adipocyte differentiation.
  • the proteins, ⁇ -casein, ⁇ -casein and ⁇ -casein are known to be one of the most nutritive groups of proteins in milk, containing all of the common amino acids and rich in essential ones.
  • ⁇ -Casein [(si) 23 kDa and (s2) 25 kDa] ⁇ -casein (24 kDa) and ⁇ -casein (19 kDa) are the most abundant proteins found in milk accounting for 80% of total protein. There have been a number of reports of the bioactive components from the casein proteins.
  • bioactive peptides from ⁇ -casein and ⁇ -casein peptide fragments have recorded antiproliferative, pro-apoptotic, ACE-inhibitor, immunomodulatory, opioid antagonistic and pro-proliferative properties
  • K -casein bioactive peptides have been shown to be anti-thrombotic and opioid antagonistic (Meisel, Curr Med Chem. 12(16): 1905-1919, 2005; Meisel and Bockelmann, Antonie van Leeuwenhoek 76(l-4) ⁇ 201- 215, 1999).
  • Caseins are a slow-digesting source of amino acids as opposed to the fast- - A -
  • Zemel et al (US Patent Application No. 2004/0197382) have proposed inducing the loss of adipose tissue by providing a high calcium diet.
  • the high calcium is provided in the form of dairy products.
  • Ward et al (US Patent Application No. 2003/0165574) suggest that a mixture of milk minerals plus a protein source (a specific casein fragment) may cause weight loss.
  • the casein fragment is proposed to act on gastrointestinal release of CCK to modulate satiety.
  • Lactoperoxidase is a member of the whey protein family, the second most abundant group of milk proteins. Whey constitutes 20% of total protein in milk. Lactoperoxidase is a 78 kDa heme containing oxidation-reduction enzyme present in milk and secretory glands. Lactoperoxidase consists of a single polypeptide chain of 612 amino acid residues, four or five carbohydrate chains constituting a total of approximately 10% of the molecular weight and accounts for 0.25-0.5% of total protein found in whey.
  • Lactoperoxidase is a highly useful protein, as it has anti-microbial activity which permits its use as a fungicide, viricide, protozoacide and bacteriocide both in products which need preservatives and in therapeutic products (Meisel, 2005 supra; Marshall, Altern Med Rev. 9(2): 136- 156, 2004).
  • bovine lactoperoxidase is known to catalyze the oxidation of a number of organic molecules such as thiols, phenols, catecholamines, steroid hormones, halides and nitrite (Tanaka et al, Biosci. Biotecnol. Biochem.
  • lactoperoxidase nor a fragment or fragments of lactoperoxidase or purified milk fractions containing lactoperoxidase has been shown to inhibit adipocyte differentiation or contribute to control of obesity.
  • Whey is a collection of globular proteins that result as a by-product from cheese production. Whey is known to have bioactive compounds and has known nutritional benefits.
  • Whey In milk, there are a number of glycoproteins which include lactoferrin, secretory IgA, IgG, IgM, free secretory component (FSC), and milk mucin.
  • Glycoproteins isolated from mammals have lipid mobilizing properties as outlined by Tisdale and Todorov (WO/1999/062939) characterizing Zn- ⁇ 2-glycoprotein as a therapeutic derived from a murine tumor to possibly control obesity.
  • Kodama and Kimura US Patent No.
  • the present invention is predicated in part on the identification of regulators of adipogenesis from milk fractions including whey.
  • the regulators are, in particular, inhibitors of adipogenesis.
  • the identification of the adipogenic inhibitors enables a nutraceutical and/or therapeutic approach to controlling obesity and complications arising therefrom including diabetes, hypertension and heart disease.
  • the inhibitors identified from milk or whey comprise one or more glycoproteins from or a fraction comprising glycoproteins or crypteins or muteins of any of these glycoproteins.
  • Purified milk fractions are also provided which comprise inhibitors of adipogenesis, the fractions being those which have ⁇ -casein, ⁇ -casein or lactoperoxidase.
  • Mimetics of these regulators of adipogenesis are also contemplated herein.
  • a method for production of a specific glycoprotein fraction isolated from whey is provided that has the ability to inhibit adipocyte differentiation and lipid accumulation as well as inhibiting or reducing weight gain and detrimental changes in metabolic parameters associated with a high fat diet. It is proposed to utilize the adipogenic inhibitors in nutraceutical and pharmaceutical formulations, as food additives and supplements and as targets for the generation of mimetics.
  • a method for isolating the bioactive glycoprotein fraction from whey and other sources is also provided, the glycoproteins having adipogenic controlling properties
  • one aspect herein contemplates a method for inhibiting adipogenesis or a process associated with adipogenesis in a subject, the method comprising administering to the subject an effective amount of an adipogenesis regulator, the regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these proteins or is a milk- derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • an adipogenesis regulator the regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these proteins or is a milk- derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • Another aspect of the present invention provides a method for treating or preventing obesity or a complication arising therefrom in a subject, the method comprising administering to the subject an effective amount of an adipogenesis regulator, the regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • an adipogenesis regulator the regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • Still another aspect of the present invention is directed to the use of a regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or is a milk- derived fraction containing ⁇ -casein, ⁇ -casein and/or lactoperoxidase in the manufacture of a nutraceutical, or food supplement to control obesity in a subject.
  • a regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or is a milk- derived fraction containing ⁇ -casein, ⁇ -casein and/or lactoperoxidase in the manufacture of a nutraceutical, or food supplement to control obesity in a subject.
  • a regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or is a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase in the manufacture of a medicament to control obesity in a subject.
  • a nutraceutical formulation comprising a regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • a therapeutic composition when used in the treatment of obesity or a complication arising therefrom in a subject comprising a regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase, the composition further comprising one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • Even yet another aspect of the present invention is directed to a food additive or supplement which reduces adipogenesis in a subject, the additive or supplement comprising a regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • a regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • a further aspect of the present invention relates to a method for enriching whey for glycoproteins and proteins, the method comprising subjecting the whey to a single multi- lectin fractionation, the lectins selected from concanavalin A, jacalin and wheat germ agglutinin or at least one other lectin substituting for concanavalin A , jacalin or wheat germ agglutinin and then screening a fraction or isolated molecule resulting therefrom for a desired activity such as adipogenesis regulating activity.
  • formulations may be administered in a number of convenient ways such as by oral, subcutaneous, intravenous or rectal administration.
  • Figure 1 is a photographic representation of SDS-PAGE analysis of bovine milk- derived whey and MLAC whey glycoprotein fraction. Lane 1) Molecular weight markers; Lane 2) 20 ⁇ g whey; Lane 3) 20 ⁇ g MLAC whey glycoprotein fraction eluted with acetic acid. (Abbreviations MLAC whey, glycoprotein fraction from whey).
  • Figure 2 is a photographic representation showing adipogenesis assay data following treatment of cells with dairy-derived protein preparations AO to HO and cryptein library D5.
  • AO to HO represent chromatographically purified preparations of major bovine milk-derived proteins ranging in purity from approximately 70-90%). All libraries are assayed at a final concentration of 0.5 mg/ml.
  • A ⁇ -lactalbumin
  • B ⁇ - lactoglobulin
  • C lactoferrin
  • D lactoperoxidase
  • E ⁇ -casein
  • F ⁇ -casein
  • G ⁇ -casein and H, whey protein; 0, native protein; 5, 2 hr peptic digest
  • MDI combination of methylisobutylxanthine, dexamethsaone and insulin
  • FIG. 3 is a graphical representation of adipogenesis assay data following treatment of cells with various concentrations of a purified MLAC whey fraction. Adipocyte differentiation and lipid accumulation was quantitatively measured using Nile Red fluorescence. (Abbreviations: MDI, combination of methylisobutylxanthine dexamethasome and insulin; MLW, glycoprotein fraction from whey; TNF, tumour necrosis factor). The MLW fraction is a potent inhibitor of adipogenesis over a broad concentration range.
  • MDI combination of methylisobutylxanthine dexamethasome and insulin
  • MLW glycoprotein fraction from whey
  • TNF tumour necrosis factor
  • FIG 4 is a photographic representation of a Western blot analysis of the key adipocyte transcription factor PPAR ⁇ .
  • Preadipocyte cultures were differentiated in the presence of an MLAC whey preparation at a final concentration of 300, 100 and 33 ⁇ g/ml.
  • Con control
  • MDI combination of methylisobutylxanthine dexamethasome and insulin
  • MLAC whey glycoprotein fraction from whey
  • PPAR ⁇ peroxisome proliferator-activated receptor ⁇
  • TNF- ⁇ tumour necrosis factor- ⁇
  • Figure 5 is a graphical representation of the effects of low and high dose of LAPOOl administration in a high fat diet animal model.
  • Rats fed a high fat diet over 28 days were treated by constant subcutaneous infusion via mini-pumps with either Vehicle, low dose LAPOOl (0.5 mg/rat/day) or high dose LAPOOl (2.5 mg/rat/day).
  • A) mean weekly body weight (grams) over 4 weeks; B) mean change in body weight (grams) (change from baseline to day 28); C) mean change in blood glucose levels (mmol/L) (from baseline to day 28); D) mean plasma insulin levels (ng/mL); E) mean plasma adiponectin levels (ng/mL) and F) mean plasma leptin levels (ng/mL) in low dose (0.5 mg/rat/day) and high dose (5 mg/rat/day) LAPOOl treated Sprague Dawley rats fed a high fat diet compared to vehicle (n 10 rats per group).
  • a glycoprotein includes a single glycoprotein, as well as two or more glycoproteins
  • an adipogenesis inhibitor includes a single adipogenesis inhibitor, as well as two or more adipogenesis inhibitors
  • reference to “the invention” includes a single or multiple aspects of an invention, and so forth.
  • Embodiments disclosed and described herein relate to the identification of proteins, including glycoproteins and crypteins and muteins thereof and fractions comprising one or more proteins, including glycoproteins and/or crypteins or muteins thereof from milk or milk product which inhibit adipogenesis in a subject.
  • Alternative sources of the proteins are also contemplated herein (e.g. soy milk).
  • Particular fractions are whey-derived glycoprotein fractions generated by a single multi-lectin fractionation and which exhibit the antiadipogenic activity.
  • Other fractions include milk fractions which can be defined as a fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase, even if these proteins are not themselves the bioactive glycoprotein.
  • Reference to "inhibit" in relation to adipogenesis includes retarding or otherwise delaying or reducing adipogenesis as well as processes associated with adipogenesis.
  • the inhibition may be transient or permanent and may be total inhibition or partial inhibition.
  • Adipogenesis includes the differentiation of pre-adipocytes into mature adipocytes.
  • reference to inhibition of adipogenesis can be considered, in one embodiment, as the inhibition of the differentiation process. This can be measured ex vivo from a subject or in an in vitro assay.
  • embodiments herein encompass the inhibition of lipogenesis which is a metabolic process of fat deposit formation.
  • inhibition of a process associated with adipogenesis includes lipogenesis.
  • one aspect herein contemplates a method for inhibiting adipogenesis or a process associated with adipogenesis in a subject, the method comprising administering to the subject an effective amount of an adipogenesis regulator, the regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one of these glycoproteins or a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • an adipogenesis regulator the regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one of these glycoproteins or a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • Milk or a milk by-product or whey is subjected to fractionation and/or separation means to generate groups of proteins or single proteins that have an inhibitory effect on adipogenesis in an in vitro screening assay.
  • the fractionation and separation processes includes, but not limited to, ion exchange chromatography, size-based chromatography, affinity-binding chromatography including lectin-binding and immunoaffinity chromatography as well as protease-based or chemical-based fractionation and separation to generate peptides referred to herein as "crypteins”.
  • the generation of crypteins is disclosed in International Publication No. 2004/008148 and reviewed by Autelitano et al, Drug Discov. Today 11 (7/8) :306-314,2006 which is incorporated herein by reference.
  • Reference to a "whey fraction” includes the fraction of whey which comprises one or more glycoproteins which are bioactive in an adipogenesis inhibition assay.
  • the present invention from a milk fraction or product which comprises one or more of ⁇ -casein and/or ⁇ -casein and/or lactoperoxidase such milk fractions or products are proposed to contain the bioactive molecule of the present invention.
  • the bioactive molecule is not necessarily one of ⁇ -casein, ⁇ -casein or lactoperoxidase.
  • the proteins or peptides or fractions or crypteins obtained may also be subjected to random or site-directed mutagenesis to generate mutated molecules referred to herein as "muteins".
  • a "protein” herein includes a "glycoprotein”. The protein may also be more commonly regarded as a peptide (or glycopeptide).
  • bioactive molecules are identified on the basis of an in vitro assay of adipogenesis and are referred to herein as "adipogenesis regulators", “adipogenesis modulators”, “adipogenesis inhibitors”, “adipogenesis medicaments” or other like terms. Notwithstanding the adipogenesis regulators may also have other biological activities such as inhibiting lipogenesis.
  • a method for identifying an adipogenesis regulator from whey or milk or a milk product comprising providing a library of proteins, protein- containing fractions, crypteins and/or muteins from the whey or milk or milk product and screening the library for an ability to inhibit adipogenesis in an in vitro assay, optionally subjecting protein or cryptein or mutein fractions to separation means to identify particular molecules or groups of molecules or sub-fractions which inhibit adipogenesis and then identifying the adipogenesis regulator as an isolated protein, sub-fraction, cryptein or mutein.
  • the library is generated by fractionation of whey or milk or a milk products by any one of a number of chromatographic and molecule-separating methods.
  • Particular methods include ion exchange chromatography, affinity chromatography such as immunoaffinity chromatography as well as lectin-based affinity chromatography.
  • the whey is subjected to lectin-based affinity chromatography comprising three lectins, concanavalin A, jacalin and wheat germ agglutinin (Yang and Hancock, J Chromatography A 1053: 79-88, 2004) and whey- derived glycoproteins are concentrated using a single step elution with 0.1M acetic acid.
  • any one or more of the above-listed lectins may be replaced by another lectin.
  • Other lectins which may be employed include peanut lectin (PNA), lentil lectin (LCA), Lens culinaris agglutinin (LCA), Griffonia (Bandeiraea) simplicifolia lectin II (GSLII), Aleuria aurantia lectin (AAL), Hippeastrum hybrid lectin (HHL 5 AL), Sambucus nigra lectin (SNA,EBL), Maackia amurensis lectin II (MAL II), Ulex europaeus agglutinin I (UEA I), Lotus tetragonolobus lectin (LTL), Galanthus nivalis lectin (GNL), Euonymus europaeus lectin (EEL) and Ricinus communis agglutinin I (RCA).
  • PNA peanut lectin
  • LCDA lentil lectin
  • LCDA Lens
  • the assay to identify the adipogenesis regulators comprises subjecting a protein source such as but not limited to whey or milk or milk product to digestion, cleavage and/or reduction to generate a series of digested or reduced fractions, subjecting each fraction to an adipogenesis assay to identify fractions having adipogenesis regulating activity, and then optionally subjecting the active fractions to separation or purification means to generate single proteins, crypteins, muteins or enriched sub-fractions having adipogenesis regulator activity.
  • an adipogenesis regulator includes, in one embodiment, a regulator of lipogenesis.
  • protein as used herein shall be taken to refer to any polymer consisting of amino acids linked by covalent bonds and this term includes within its scope parts or fragments of full length proteins, such as, for example, polypeptides, peptides and shorter peptide sequences consisting of at least two amino acids, more particularly at least about 5 amino acid residues.
  • protein includes all moieties containing one or more amino acids linked by a peptide bond.
  • this term includes within its ambit polymers of modified amino acids, including amino acids which have been post- translationally modified, for example by chemical modification including but not restricted to glycosylation, phosphorylation, acetylation and/or sulphation reactions that effectively alter the basic peptide backbone.
  • a protein herein may be derived from a naturally- occurring protein, and in particular may be derived from a full-length protein by chemical or enzymatic cleavage, using reagents such as CNBr, or proteases such as trypsin or chymotrypsin, amongst others.
  • reagents such as CNBr, or proteases such as trypsin or chymotrypsin, amongst others.
  • such peptides may be derived by chemical synthesis using well known peptide synthetic methods.
  • the proteins are isolated following, for example, ion exchange or affinity chromatography.
  • amino acid sequence variants referred to herein as "muteins"
  • mutants amino acid sequence variants
  • amino acid substitutions may contain one or more amino acid substitutions, deletions, or insertions in a naturally-occurring amino acid sequence.
  • Such muteins may be synthesized by chemical peptide synthesis.
  • Amino acid substitution reactions are well-known in the art. Rules for making such substitutions are well known. More specifically, conservative amino acid substitutions are those that generally take place within a family of amino acids that are related in their side chains.
  • muteins which have undergone unnatural modifications such as but not limited to protection, carboxylation, and derivatization by amide and non-amide bonds as well as covalent and non-covalent modification.
  • protein also includes recombinantly generated polypeptides, oligopeptides or shorter peptide sequences. Protein which is produced in vitro or in a host cell by the expression of a genetic sequence encoding the protein, which genetic sequence is under the control of a suitable promoter, wherein a genetic manipulation has been performed in order to achieve said expression. Accordingly, the term “recombinant protein "clearly encompasses proteins produced by the expression of genetic sequences contained in viral vectors, cosmids or plasmids that have been introduced into prokaryotic or eukaryotic cells, tissues or organs.
  • Genetic manipulations which may be used in this context will be known to those skilled in the art and include, but are not limited to, nucleic acid isolation, restriction endonuclease digestion, exonuclease digestion, end-filling using the Klenow fragment of E. coli DNA polymerase I to T4 DNA polymerase enzymes, blunt-ending of DNA molecules using T4 DNA polymerase or ExoIII enzymes, site- directed mutagenesis, ligation, and amplification reaction.
  • the initial library of proteins comprises a heterogeneous and unfractionated mixture of proteins derived from a precursor protein source (or protein mixture or protein-containing biological extract) such as whey or milk which provides a comprehensive range of potentially bioactive proteins or crypteins or muteins.
  • a precursor protein source or protein mixture or protein-containing biological extract
  • whey or milk which provides a comprehensive range of potentially bioactive proteins or crypteins or muteins.
  • the library is conveniently subjected to initial analysis or characterization to provide information on the activity or size or other characteristics of the component proteins, for example by adipogenesis assay or matrix assisted laser desorption time of flight mass spectrometry (MALDI-ToF MS).
  • initial analysis or characterization to provide information on the activity or size or other characteristics of the component proteins, for example by adipogenesis assay or matrix assisted laser desorption time of flight mass spectrometry (MALDI-ToF MS).
  • Initial screening of the library to confirm that it includes bioactive proteins may particularly be carried out using an in vitro adipogenesis assay.
  • the library After the library has been confirmed as including bioactive proteins, it is fractionated by suitable means of fractionation including but not limited to chromatographic methods such as, but not limited to, ion exchange, size exclusion, hydrophobic interaction and/or reverse phase-high performance liquid chromatography, field-flow fractionation (including but not limited to sedimentation, flow, thermal and steric), and electrophoresis in order to provide fractions of the library for subsequent further screening.
  • chromatographic methods such as, but not limited to, ion exchange, size exclusion, hydrophobic interaction and/or reverse phase-high performance liquid chromatography, field-flow fractionation (including but not limited to sedimentation, flow, thermal and steric), and electrophoresis in order to provide fractions of the library for subsequent further screening.
  • field-flow fractionation including but not limited to sedimentation, flow, thermal and steric
  • electrophoresis in order to provide fractions of the library for subsequent further screening.
  • This further screening may be carried out by any suitable screening assay or assays as
  • each fraction may, if desired, be subjected to one or more further cycles of fractionation by suitable means of fractionation including but not limited to chromatography, field-flow fractionation (including but not limited to sedimentation, flow, thermal and steric), and electrophoresis to form sub-fractions, followed by screening of each sub-fraction as described above so as to identify an active sub-fraction or active sub-fractions which include bioactive proteins (i.e. sub-fractions or proteins which have adipogenesis regulator activity).
  • suitable means of fractionation including but not limited to chromatography, field-flow fractionation (including but not limited to sedimentation, flow, thermal and steric), and electrophoresis to form sub-fractions, followed by screening of each sub-fraction as described above so as to identify an active sub-fraction or active sub-fractions which include bioactive proteins (i.e. sub-fractions or proteins which have adipogenesis regulator activity).
  • Each fraction or sub-fraction which is produced may also be subjected to analysis or characterization as described above, for example by adipogenesis assay or MALDI-ToF MS, so as to provide information on the activity, size or other characteristics of the component proteins in the fraction or sub-fraction.
  • milk-derived fractions containing ⁇ -casein, ⁇ -casein and lactoperoxidase as well as crypteins and muteins thereof and fractions comprising same and whey glycoproteins and fractions comprising same as well as crypteins and muteins of the whey glycoproteins are proposed to be adipogenesis regulators useful in the inhibition of adipogenesis.
  • regulators of adipogenesis include, in an embodiment, regulators of lipogenesis or other metabolic processes associated with obesity.
  • the source of the milk is not critical to the practice of the embodiments herein and may be from any mammal including a human, cow, sheep, goat, horse, pig, camel, laboratory test animal (e.g. mouse, rat, rabbit, guinea pig), companion animal (e.g. dog, cat) or captive wild animal (e.g. elephant, zebra, kangaroo).
  • milk includes soy milk and other legume milks.
  • another aspect of the present invention provides a method for treating or preventing obesity or a complication arising therefrom in a subject, the method comprising administering to the subject an effective amount of an adipogenesis regulator, the regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • an adipogenesis regulator the regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • a regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • the "subject" as used herein refers to an animal, particularly a mammal and more particularly a primate including a lower primate and even more particularly a human who can benefit from the formulations and methods of the present invention.
  • a subject regardless of whether a human or non-human animal may be referred to as a subject, an individual, patient, animal, host or recipient.
  • the formulations and methods of the present invention have applications in human medicine, veterinary medicine as well as in general, domestic or wild animal husbandry. Hence, the formulations described herein are applicable for the treatment of humans, livestock animals, racing industry animals and companion animals.
  • non-human primates such as marmosets, baboons, orangutangs, lower primates such as tupia, livestock animals, laboratory test animals, companion animals or captive wild animals.
  • a companion animal includes a dog and cat.
  • a human is the most preferred target.
  • non-human animal models may be used.
  • Examples of laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model as do primates and lower primates. Livestock animals include sheep, cows, pigs, goats, horses and donkeys. Racing industry animals include horses, camels and dogs.
  • the adipogenic regulators herein may be formulated from any convenient manner such as in a nutraceutical formulation or a pharmaceutical formulation using standard formulation technology.
  • a nutraceutical formulation comprising a regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • a therapeutic composition when used in the treatment of obesity or a complication arising therefrom in a subject comprising a regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or a milk- derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase, the composition further comprising one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • a regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or a milk- derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase
  • the composition further comprising one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • Even yet another aspect of the present invention is directed to a food additive or supplement which reduces adipogenesis in a subject, the additive or supplement comprising a regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • a regulator of adipogenesis being a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • Embodiments herein further contemplate a method of isolating a glycoprotein or fraction of glycoproteins from milk, the method comprising: (a) contacting a composition, comprising at least, but not limited to, three different lectins attached to a solid support, with a sample containing the glycoproteins, under conditions that promote binding of glycoproteins in the sample to the lectins, thereby providing a bound sample;
  • the lectins are concanavalin A, jacalin and wheat germ agglutinin.
  • Kits are contemplated herein such as comprising compartments or containers each containing a glycoprotein from whey or a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins or a milk- derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • the kits may be packaged for sale as food additives or supplements or may be in the form of nutraceutical or pharmaceutical packs.
  • the proteins may be in a freeze dried form, the contents of which may be reconstituted prior to use in food or as a medicament.
  • the present invention further provides a method for inhibiting adipogenesis or a process associated with adipogenesis in a subject, the method comprising administering to the subject an effective amount of an adipogenesis regulator, the regulator of adipogenesis selected from the list consisting of a glycoprotein from whey and a whey fraction comprising one or more glycoproteins or a cryptein or mutein of one or more of these glycoproteins.
  • Yet another aspect of the present invention provides a method for inhibiting adipogenesis or a process associated with adipogenesis in a subject, the method comprising administering to the subject an effective amount of an adipogenesis regulator, the regulator consisting of a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • an adipogenesis regulator consisting of a milk-derived fraction comprising ⁇ -casein, ⁇ -casein and/or lactoperoxidase.
  • formulations described herein are also contemplated when used for the treatment of obesity or complications arising therefrom such as hypertension, diabetes or heart disease.
  • Administration of the formulations of the present invention may be any convenient route such as by oral, subcutaneous and subcutaneous mini-pump, intravenous or rectal administration.
  • Formulations suitable for oral, subcutaneous, intravenous or rectal administration are referred to as oral, subcutaneous, intravenous or rectal formulation.
  • Figure 1 shows SDS-PAGE analysis of bovine milk whey and whey glycoproteins (MLAC whey) samples against molecular weight samples.
  • SDS-PAGE was performed using 4-12% w/v Bis-Tris gels on a Novex NuPAGE system.
  • the proteins are resolved with the NuPAGE MOPS SDS running buffer in a Novex Mini-Cell system at 200 volts.
  • the proteins are visualized by staining with SimplyBlue safe stain blue.
  • MLAC whey is a fraction of whey glycoprotein obtained following multi-lectin chromatography enrichment using lectins concanavalin A, jacalin and wheat germ agglutinin.
  • the bound glycoproteins were eluted with acetic acid (Lane 3) to provide an enriched whey glycoprotein fraction.
  • the eluted glycoproteins range in molecular weight from less than 14 kDa to greater than 100 kDa. There is clear visible enrichment of glycoproteins (Lane 3) using multi-lectin chromatography when compared to whey starting material (Lane 2).
  • Preadipocytes were routinely passaged in Growth Medium (DMEM, 10% v/v bovine calf serum, penicillin, streptomycin, amphotericin B) and were plated in a volume of 100 ⁇ l in this medium in 96 well microplates at a density of 10,000 cells per well for assay. Preadipocytes were grown at 37 0 C for 3 days in a humidified atmosphere containing 5% CO 2 to reach a post- confluent state.
  • Growth Medium DMEM, 10% v/v bovine calf serum, penicillin, streptomycin, amphotericin B
  • Dairy protein-derived compounds were also added concurrently with Induction Medium to test their effects on modulating differentiation of preadipocytes to mature, lipid-laden adipocytes.
  • Assay plates were routinely allowed to differentiate for 6-7 days, during which time wells were monitored by phase contrast microscopy to document the altered cellular phenotype and accumulation of refractive lipid vesicles.
  • Medium was then removed from the assay plates and cells were fixed in 1% v/v paraformaldehyde in phosphate buffered saline for 20 minutes at room temperature. The fixative was aspirated, wells were washed twice with 200 ⁇ l PBS and prepared for Oil Red O staining using reagents from an Adipogenesis Kit (Chemicon).
  • MLAC whey glycoprotein fraction prepared as described in Example 1 was tested for its ability to modulate adipogenesis in an in vitro assay and it was found that this fraction highly enriches the anti-adipogenesis bioactivity (Figure 3).
  • Figure 3 To quantify the effect of this dairy-derived fraction on the differentiation to mature adipocytes and accumulation of intracellular lipid, an identical strategy was employed to grow and treat 3T3-L1 preadipocytes as outlined in Example 2. Instead of staining lipid droplets with Oil Red O, intracellular lipid droplets were stained with the vital stain Nile Red and lipid accumulation was quantitated by measuring the subsequent fluorescence.
  • Cultures were treated with a range of concentrations of MLAC whey derived fraction from 3.9 -500 ⁇ g/ml. Following differentiation for 6-7 days as described, media was removed from wells, cells were washed with 200 ⁇ l PBS and replaced with 200 ⁇ l fresh PBS. The plate was then transferred to a Polarstar multi-mode plate reader (BMG) equipped with automatic injectors. Each well was injected with 5 ⁇ l Nile Red dye solution (AdipoRed reagent from Cambrex) followed by a 2 second shake. The plate was then incubated at room temperature for 10 minutes before reading fluorescence at Ex 485 nm / Em 590 nm.
  • BMG Polarstar multi-mode plate reader
  • Con and MDI refers to control wells treated without or with adipocyte Induction Medium containing Methyl-3-isobutylxanthine, Dexamethasone and Insulin.
  • TNF refers to 5 ng/ml TNF- ⁇ used as a control inhibitor of adipocyte differentiation.
  • Dairy-derived purified MLAC Whey fraction inhibits signalling associated with adipogenesis.
  • a purified MLAC Whey fraction that was shown to inhibit adipocyte differentiation and accumulation of lipid was further characterised by determining its effects on a major signalling cascade associated with adipogenesis (Figure 4).
  • Figure 4 To quantify the effect of partially purified MLAC Whey fraction on the induction of the key adipocyte transcription factor PPAR ⁇ , 3T3-L1 preadipocytes were grown and differentiated as outlined in Example 2 in 12 well microplates.
  • As a control for inhibition of adipocyte differentiation cells were treated with 5 ng/ml TNF- ⁇ which was added concurrently with Induction Medium.
  • MLAC Whey fractions were also added at three concentrations concurrently with Induction Medium to test their effects on modulating differentiation of preadipocytes to mature, lipid-laden adipocytes. Assay plates were allowed to differentiate for 6 days, during which time wells were monitored by phase contrast microscopy to document the altered cellular phenotype and accumulation of refractive lipid vesicles. Medium was then removed from the assay plates, cells were washed twice with ice-cold PBS and cell lysates were prepared by extracting in 120 ⁇ l ice-cold RIPA buffer.
  • Total protein content of the cell lysates was quantitated by BCA assay and 25 ⁇ g protein was loaded and separated on a 4-12% w/v NuPAGE Bis-Tris gel run in MES buffer. Following transfer to nitrocellulose, Western blotting was performed using a rabbit polyclonal anti- PPAR ⁇ IgG (Upstate) that recognises both PPAR ⁇ 1 and PPAR ⁇ 2 as well as their phosphorylated forms. Labelled immunoreactive-PPAR ⁇ bands were visualised using a chemiluminescence reagent (ECL; Amersham) and exposure to hyperfilm.
  • ECL chemiluminescence reagent
  • Dairy-Derived Extract LAPOOl is a Potent Anti-adipogenic Agent that Significantly
  • Compound LAPOOl is a specific batch of the bioactive MLAC whey preparation described in the previous Examples.
  • a scaled up version of the method described in Example 1 was used to generate gram quantities of LAPOOl to test the efficacy of this preparation in an animal model.
  • the LAPOOl enrichment process described provides a scalable technique adaptable for large scale industrial applications.
  • the acetic acid eluted LAPOOl fraction was used for all the subsequent in vitro and in vivo studies.
  • the bioactivity of LAPOOl was confirmed in vitro and it was shown that adipocyte differentiation and lipid accumulation was inhibited over a wide concentration range (3.9 to 500 ⁇ g/ml).
  • Constant subcutaneous administration was achieved via osmotic mini- pump providing continuous infusion of LAPOOl and vehicle for a period of 28 days (mini- pumps were changed half way through the study at day 14).
  • the study was designed to examine a broad range of endpoints relevant to various parameters that include: body weight prior to and throughout study; post mortem weight of organs and fat deposition; blood glucose levels at beginning and at post mortem; post mortem blood plasma insulin, leptin and adiponectin.
  • the animal study showed that LAPOOl at both the high and low dose was effective at preventing several metabolic perturbations associated with a high fat diet.
  • Figure 5 illustrates some of the significant findings of LAPOOl administration in a high fat fed animal model.
  • LAPOOl is an effective agent that can act in vivo to ameliorate detrimental effects of a high fat diet including body weight gain and related metabolic parameters such as plasma glucose levels.

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0393850B1 (de) * 1989-04-19 1993-01-07 Snow Brand Milk Products Co., Ltd. Verfahren zur Herstellung von kappa-Casein-Glykomakropeptiden
US20030165574A1 (en) * 2002-03-01 2003-09-04 Ward Loren Spencer Compositions and methods for treatment of body weight conditions
WO2004008148A1 (en) 2002-07-10 2004-01-22 Cryptome Pharmaceuticals Ltd Method for detection of bioactive peptides
US20050106218A1 (en) * 2002-03-01 2005-05-19 Glanbia Nutritionals (Ireland) Limited Compositions and methods for treatment of body weight conditions
US6929806B2 (en) * 2001-01-16 2005-08-16 Snow Brand Milk Products Co., Ltd. Agents for improving lipid metabolism and reducing high blood pressure
US20060257497A1 (en) * 2003-02-07 2006-11-16 Campina B.V. Use of Tryptophan rich peptides
US20090012001A1 (en) * 2006-06-09 2009-01-08 Morinaga Milk Industry Co., Ltd. Lipid metabolism-improving agent

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001527046A (ja) * 1997-12-30 2001-12-25 エイプラス サイエンス インベスト アーベー ガラクトシルセラミド、グルコシルセラミド、ラクトシルセラミド、並びに前糖尿病、糖尿病および／または関連合併症の予防もしくは治療に使用するためのその特定キャッチャー
GB9811465D0 (en) * 1998-05-29 1998-07-29 Tisdale Michael J Glycoproteins having lipid mobilising properties and therapeutic applications thereof
US20090166224A1 (en) * 2004-05-05 2009-07-02 Ziping Yang Multi-lectin affinity chromatography and uses thereof
JP5285213B2 (ja) * 2006-03-16 2013-09-11 株式会社ロッテ 脂肪分解促進剤
FR2900342B1 (fr) * 2006-04-27 2010-08-20 Cie Laitiere Europeenne Utilisation de l'alpha-lactalbumine pour la regulation de la glycemie

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0393850B1 (de) * 1989-04-19 1993-01-07 Snow Brand Milk Products Co., Ltd. Verfahren zur Herstellung von kappa-Casein-Glykomakropeptiden
US6929806B2 (en) * 2001-01-16 2005-08-16 Snow Brand Milk Products Co., Ltd. Agents for improving lipid metabolism and reducing high blood pressure
US20030165574A1 (en) * 2002-03-01 2003-09-04 Ward Loren Spencer Compositions and methods for treatment of body weight conditions
US20050106218A1 (en) * 2002-03-01 2005-05-19 Glanbia Nutritionals (Ireland) Limited Compositions and methods for treatment of body weight conditions
WO2004008148A1 (en) 2002-07-10 2004-01-22 Cryptome Pharmaceuticals Ltd Method for detection of bioactive peptides
US20060257497A1 (en) * 2003-02-07 2006-11-16 Campina B.V. Use of Tryptophan rich peptides
US20090012001A1 (en) * 2006-06-09 2009-01-08 Morinaga Milk Industry Co., Ltd. Lipid metabolism-improving agent

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AUTELITANO ET AL., DRUG DISCOV. TODAY, vol. 11, no. 7-8, 2006, pages 306 - 314
FRESTEDT, J. L ET AL.: "A WHEY PROTEIN SUPPLEMENT INCREASES FAT LOSS AND SPARES LEAN MUSCLE IN OBESE SUBJECTS: A RANDOMIZED HUMAN CLINICAL STUDY", NUTRITION & METABOLISM, vol. 5, 2008, pages 8, XP021038429 *
See also references of EP2318021A4 *
YANG; HANCOCK, J. CHROMATOGRAPHY A, vol. 1053, 2004, pages 79 - 88
ZEMEL, M.B ET AL.: "DIETARY CALCIUM AND DAIRY MODULATION OF ADIPOSITY AND OBESITY RISK", NUTRITION REVIEWS, vol. 62, no. 4, April 2004 (2004-04-01), pages 125 - 131, XP002539983 *

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